Somatic Diversification of Immunoglobulin Genes in Galt

描述（由申请方提供）：脊椎动物淋巴组织发育和粘膜免疫需要肠道肠道微生物群。然而，人们对这些过程发生的机制知之甚少。先前在兔中的工作已经证明，肠道肠道微生物群也是免疫前抗体库发育期间IG基因的体细胞超突变（SHM）和基因转换（GC）以及B细胞选择所需的，这两者都发生在肠道相关淋巴组织（GALT）中。通过将选择的细菌菌株引入无菌GALT，我们发现一些但不是所有的微生物都具有诱导GALT发育和IG基因的SHM和GC的能力。我们鉴定了两种肠道微生物，B。fragilis和B.枯草杆菌，一起，诱导这些过程;然而，单独引入，没有生物体诱导这些过程。本研究的目的是确定这些细菌诱导GALT发育和IG基因的SHM和GC的机制，以及为什么需要两种细菌。第一个具体目标是确定细菌如何进入宿主，以及卵泡诱导细菌和非卵泡诱导细菌的差异是否在于它们如何穿过GALT的上皮。测试这一点的实验包括将细菌引入无菌阑尾，然后进行原位杂交和免疫荧光，以确定细菌位于哪些细胞中。下一个具体的目标，通过使用重组腺病毒表达的可溶性受体，将调查树突状细胞（DC）是否有助于GALT的发展和IG基因的多样化，通过分泌的B细胞生长因子，BAFF，或通过抑制DC在体内的跨上皮迁移。在目的3中，将通过用可溶性CTLA 4抑制T细胞活化和用可溶性CD 40抑制B-T细胞相互作用来研究对T细胞帮助的需要，两者都在重组腺病毒中产生。在目的4中，设计实验以寻找诱导GALT发育和一级抗体库的体细胞多样化的B细胞超抗原。这些实验很重要，因为肠腔含有的细菌细胞比人体所有其他细胞的总和多10倍，但我们对这些生物体如何促进人类健康和过敏和炎症性肠病等疾病知之甚少。

DESCRIPTION (provided by applicant): The intestinal commensal microbiota is required for development of lymphoid tissues and mucosal immunity in vertebrates. However, little is known of the mechanism by which these processes occur. Previous work in rabbits has demonstrated that the intestinal commensal microbiota is also required for somatic hypermutation (SHM) and gene conversion (GC) of Ig genes during development of the preimmune antibody repertoire, and for B cell selection, both of which occur in gut-associated lymphoid tissues (GALT). By introducing select bacterial strains into sterile GALT, we found that some but not all commensal organisms have the capacity to induce GALT development and SHM and GC of Ig genes. We identified two commensal microorganisms, B. fragilis and B. subtilis, that together, induced these processes; however, introduced singly, neither organism induced these processes. The goal of this study is to determine the mechanism by which these bacteria induce GALT development and SHM and GC of Ig genes, and why two bacteria are required. The first specific aim is to determine how bacteria enter the host, and if the difference in follicle-inducing bacteria and non-follicle-inducing bacteria resides in how they are taken up across the epithelium of GALT. Experiments to test this include introducing bacteria into germfree appendix and then performing in situ hybridization and immunofluorescence to determine in which cells the bacteria are localized. The next specific aim, by using soluble receptors expressed in recombinant adenovirus, will investigate whether dendritic cells (DC) contribute to GALT development and Ig gene diversification by secretion of the B cell growth factor, BAFF, or by inhibiting the transepithelial migration of DC in vivo. In Aim 3, the requirement for T cell help will be investigated by inhibiting the activation of T cells with soluble CTLA4 and B-T cell interactions with soluble CD40, both produced in recombinant adenovirus. In Aim 4, experiments are designed to search for a B cell superantigen that induces GALT development and somatic diversification of the primary antibody repertoire. These experiments are important because the lumen of the intestine contains 10X more bacterial cells than all other cells of the human body combined, and yet we know little about how these organisms contribute to human health and diseases such as allergy and inflammatory bowel disease.